Low molecular weight heparins (LMWHs), a commonly used anticoagulant drug in the hospitals, are carbohydrate-based molecules. Heparin is isolated from porcine intestine through a long and unreliable supply chain. LMWH is a depolymerized product of heparin. A worldwide distribution of contaminated heparin in 2007, impacting the purity and safety of LMWHs, was associated with 85 deaths in the US. This crisis revealed the vulnerability of the LMWH supply chain to adulteration. Very recently, US congress expressed concerns over the safety of heparin supply chains and urged US Food and Drug Administration to increases the efforts to eliminate the risk of contamination and adulteration. Increasing oversight on the quality control of animal-sourced heparin does not provide an alternative source for this life-saving drug if a sudden interruption of heparin supply chain happens. One ultimate solution to eliminate the problems associated with the animal- sourced heparin is to develop a cost-effective method to prepare synthetic heparin. LMWHs are complex mixtures, having average molecular masses of 3500-6000 Daltons, corresponding to 12-20 saccharide units. The efficient preparation of a synthetic LMWH could improve the safety, availability and efficacy. However, the production of homogeneous LMWHs has not been possible due to difficulties in the chemical synthesis. Glycan Therapeutics proposes to assess an innovative chemoenzymatic approach that would provide an unprecedented efficiency in the preparation of heparin. Our product will be animal-free, structurally homogenous, safe for renal-impaired patients, and have the ability of its anticoagulant activity to be neutralized by protamine. To demonstrate the scalability, a gram-scale synthesis of a new synthetic LMWH construct was completed. A series of biological, pharmacological and toxicity studies of the synthetic LMWH were conducted to justify further development. In the Phase I STTR studies proposed here, we will assess the feasibility of a pilot scale synthesis (20 grams) and the purity of the product. Aim 1 will develop scalable methods to synthesize two sugar nucleotide cofactors and one sulfo donor cofactor. Aim 2 will demonstrate the synthesis of a synthetic LMWH construct in the scale of 20 grams. In the Phase II STTR studies, we plan to optimize production protocols to scale synthesis to 200 grams for toxicology and anticoagulant efficacy studies leading to an IND. The completion of this project will modernize this century-old drug with improved safety and anticoagulant properties.